NEUROBEHAVIORAL AND NEUROENDOCRINE ASSESSMENT OF RATS
PERINATALLY EXPOSED TO POLYCHLORINATED BIPHENYLS:
A POSSIBLE MODEL FOR AUTISM
Dena N. Krishnan
A Thesis
Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE
August 2007
Committee:
Lee A. Meserve, Advisor
Howard Casey Cromwell
Mike Geusz
Luke Tsai ii
ABSTRACT
Lee A. Meserve, Advisor
Several studies have shown that perinatal exposure to xenobiotic mixtures such as
polychlorinated biphenyls (PCBs) can cause physiological and behavioral disruption.
These studies demonstrate that PCB-exposed rats are a possible model for understanding
motor and social deficits in childhood developmental disorders like autism spectrum
disorder (ASD). The mixture PCB 47/77 at 0 ppm, 12.5 ppm, or 25 ppm (w/w) of the diet
was fed to pregnant Sprague-Dawley rats impacting offspring both indirectly and directly
from GD 1 to PND 21. Motor functioning of offspring was tested at PND 14-16, 28-32,
and 60-64 with measures of general motor activity and stereotypic repetitive behavior.
Subsequently, t-maze learning acquisition and reversal was tested in males 25-29 days
old and circulating levels of the hormone vasopressin were measured at 29 days of age by
enzyme immunoassay. Numerous motoric impairments were found in PCB-exposed rats
compared to controls including: 1) altered rates of activity, 2) significant delay in the formation of grooming chain syntax, 3) a longer latency in pup righting reflex, and 4) depressed ability to complete the hang and negative geotaxis tests at various stages of development. PCB-exposed rats showed a significant delay in t-maze learning aquisition and reversal in a dose dependent manner relative to controls. These consequences likely stem from neuroendocrine disruption despite no change in systemic circulating vasopressin concentration. The different results in animals given 12.5 ppm and 25 ppm
PCB suggest use of this animal model to reveal a range of motoric disruption similar to the broad autistic phenotype.
iii
DEDICATION
To all the challenges and disappointments that make success worth striving for.
iv
ACKNOWLEDGEMENTS
First and foremost, I would like to thank my advisor, Dr. Lee Meserve, for
helping me grow as a graduate student, teacher, and researcher. It is by his example,
support, encouragement, and sense of humor that I have developed a true passion for
neuroendocrine research. I thank my other committee members: Dr. Howard Casey
Cromwell for inspiring my behavioral work, Dr. Mike Geusz for his insightful
suggestions, and Dr. Luke Tsai for his advice in making appropriate clinical connections.
I would like to acknowledge my fellow lab associates, Banafsheh Jolous-
Jamshidi, Avanti Desai, and Christina Asbrock for their companionship; and to the many undergraduate scholars, especially Trang Tran and Travis Beckwith for their meticulous involvement with the collection and scoring of my behavioral data. I wish to thank Matt
Hoostal because without his coaching and expertise, I would still be grappling with my statistics.
I extend my sincerest gratitude to the faculty, staff, and graduate students of the
Department of Biological Sciences for making my time at Bowling Green State
University so enjoyable. In particular, I must thank Lorraine DeVenney for making me
an offer I could not refuse and will never forget.
Lastly, I acknowledge my parents, family, and friends near and far for their love
and support. I especially want to thank my brothers: Josh for teaching me more about
neuroscience than I ever wanted and Anoop for being my number one fan. Thank you all
for enriching my life and helping me achieve this important goal.
v
TABLE OF CONTENTS
INTRODUCTION………………………………………………….....…………………1
MATERIAL AND METHODS……………………………………………………...….15
RESULTS
NEUROBEHAVIORAL ASSESSMENT:
Developmental Motor Activity: Open Field………………………………………………………..24 Hang Test…………...……………………………………………26 Negative Geotaxis Response……………………………………..27 Righting Reflex…….……………….……………………………29
Stereotypic Repetitive Behavior: Grooming………………………………………………………...30 T-maze Acquisition and Reversal…………………………….….34
Summary of Behavioral Results………………………………………....37
NEUROENDOCRINE STATUS: Circulating Vasopressin Concentrations………………………………....38
DISCUSSION………………………………………..…………………………………..39
REFERENCES……………….……………………………………………...…………..52
APPENDICES……………………………………………………………………...……61
vi
LIST OF FIGURES
Figure Page
1. Conceptual Idea of Term “Autism Spectrum Disorder” (ASD)……..….…….1
2. Structure of Polychlorinated Biphenyl (PCB) Molecule………..….…………5
3. The Neurohypophysis……………………………….……………...………..11
4. Choreographed “Syntactic Chain” Sequence of Grooming Actions………...19
5. Effect of PCB on Mean Horizontal Movement during Development……….24
6. Effect of PCB on Mean Rearing during Development………………………25
7. Effect of PCB on Development Mean Rear Time.……………………….….25
8. Effect of PCB on Development Mean Hang Test…………………..….…….27
9. Effect of PCB on Developmental Mean Negative Geotaxis Response……...28
10. Effect of PCB on Developmental Mean Righting Reflex.. ………….………29
11. Mean Total Grooming/ Rat ………………………………………………….31
12. Mean Chains Initiated/ Rat…………………………………………………..31
13. Percent Complete Perfect Chains ……………………………………………32
14. Percent Incomplete Chains…………………………………………………..32
15. Percent Complete Imperfect Chains…………………………………………33
16. Effect of PCB on Mean T-maze Acquisition in Juvenile male rats………….34
17. Effect of PCB on Mean T-maze Reversal Learning in Juvenile male rats…..36
18. Effect of PCB 47/77 on Mean Circulating Serum Vasopressin Concentrations in 29-Day Old Male Rats. ……………………………………………………….38
vii
LIST OF TABLES
Table No. Page 1. Timeline for first set of litters tested…………………………………….…...16
2. Timeline for second set of litters tested……………………………….……..16
3. Sample Size for all Open Field Parameters Tested…………………………..26
4. Sample Size for Mean Hang Score…………………………………………..27
5. Sample Size for Mean Negative Geotaxis Response………………………...28
6. Sample Size for Mean Righting Reflex……………………………………...29
7. Sample Size for all Grooming Parameters Tested…………………………...33
8. Sample Size for Mean Learning Days……………………………………….34
9. % Rats Meeting Acquisition Criteria ………………………………………..35
10. Sample Size for T-maze Reversal……………………………………………36
11. Summary of Behavioral Results in Rats Exposed to PCB 47/77 at
0, 12.5 and 25 ppm ……………………………………………………....37
12. Sample Size for Serum Vasopressin Analysis……………………………….38
13. Comparison of PCB Congeners/ Doses and Motor Outcomes……..………..40
14. PCB Exposure as a Possible Animal Model for ASD……………….………51 1
INTRODUCTION
Autism spectrum disorder (ASD) is a term used to describe a group of developmental disorders associated with substantial deficits in three specific areas: (1) reciprocal social interaction, (2) communication, and (3) the presence of repetitive and stereotyped behaviors and unusual interests (DSM-IV; American Psychiatric Association,
1994). These core clinical features generally appear before age 3 and last throughout a lifetime. ASD occurs in all racial, ethnic, and socioeconomic groups and is four times more likely to occur in boys than girls (CDC, 2007; Tsai 1999). The definition of ASD has evolved since Kanner first described the disorder in 1943 (Kanner, 1943), and now encompasses a wide range of behavioral severity with varying levels of cognitive and linguistic functioning (Tsai and Ghaziuddin, 1996). The term ASD includes the commonly recognized disorder autism, as well as Asperger’s syndrome, and pervasive developmental disorders not otherwise specified (PDDNOS). Since the etiology of ASD is unknown, the heterogeneity originally described on a continuum or “spectrum,” is presented here as different syndromes with overlapping clinical features (Figure 1).
Autism
Asperger’s PDDNOS
Figure 1: Conceptual Idea of the term “autism spectrum disorder” (ASD). (Adapted and modified from Tsai, 1999) 2
Across ASD, the social domain may range from a complete absence of interest in interacting with others to more subtle difficulties managing complex social interactions that take into account the intentions of other people (Pelphrey et al., 2004). Individuals with Asperger’s syndrome tend to be argumentative and aggressive with a condescending affect, features rarely seen in individuals with autism (Ghaziuddin et al., 1991).
Stereotypic repetitive behaviors may range from abnormal gait and preference for sameness to more complex elaborate rituals (Tsai and Ghaziuddin, 1996). Language deficits, while marked in some autistic individuals who are deemed completely nonverbal, can be mild and limited to the presence of pragmatic language deficits in higher functioning individuals with autism (Hollander, 2003; Koger et al., 2005).
Several studies have implicated environmental factors as acting synergistically with genetic factors to cause the broad autistic phenotype (Hollander, 2003). Much of this literature focuses on heavy metals such as mercury and lead and commercial chemicals such as polychlorinated biphenyls (PCB) because of their direct neurotoxic, endocrine disrupting potential (Hubbs-Tait et al., 2005). Young children often encounter higher levels of toxicants than adults due to what Weiss (2000) calls the unique “spatial ecology” of childhood. Young children spend considerably more time on the floor breathing dust, drink breast milk, and ingest more juice, fruit and water, giving them increased exposure to pesticide residues and contaminants than adults (Koger et al.,
2005). It is well established that the developing nervous system is particularly vulnerable to environmental insult (Weiss, 2000). Exposure to even minimal levels of most toxicants during critical periods of development can result in amplified, permanent developmental damage. 3
In addition to the range of severity, an apparent rise in global prevalence of ASD supports a significant role for nongenetic environmental factors (Hollander, 2003).
Although estimates on the prevalence of autism vary widely depending on the scope of the definition of the term, some recent studies using the current criteria found prevalence rates for ASD between 2 and 6 per 1,000 individuals, or 1 in 166 (CDC, 2006). Most recently (2007), the Autism and Developmental Disabilities Monitoring Network
(ADDM) of the Centers for Disease Control and Prevention (CDC) released data reporting that 1 in 150 8-year-old children in multiple areas of the United States had
ASD. As shocking as these statistics are, according to the CDC (2006), approximately
17% of children have at least some type of developmental disability that impacts cognitive funtion, language or learning ability, emotional state, sensory and motor function, and/or physical growth. When data supporting the increasing prevalence of neurodevelopmental disorders in humans are compared with those for the increase in synthetic chemical production and release, the rising curves began to merge around 1970
(Colborn, 2004). It was at this time that the first generation of humans potentially exposed in utero to synthetic chemicals began to have children.
For example, a plastic monomer, bisphenol A (BPA) was introduced in the 1920s,
PCB compounds were introduced in 1929, and DDT became available for retail sale in
1938. Although individuals were being exposed to these chemicals since the early 1920s, it was not until the end of WWII that exposure increased to such an extent that the vast numbers of adults exposed daily were accumulating significant amounts of these chemicals in their bodies. In terms of generation time, these individuals in the 1950s produced the first genreation of offspring exposed to numerous synthetic chemicals in the 4 womb and at increased levels. By 1970 these post-WWII babies were having children of their own. It was during this time that previously rare neurodevelopmental disorders began to emerge and soon became household names (Colburn, 2004).
Genetic studies support the multifactorial theory of ASD, in particular because the concordance in monozygotic (MZ) twins is less than 100% and the phenotypic expression of the disorder varies even within MZ twins. The concordance rate for MZ twins is 36-
91% compared to either no concordance or 10% concordance in dizygotic (DZ) twins, depending on whether the phenotype is narrowly or broadly defined (Bailey et al., 1995;
Steffenburg et al., 1989). Family studies yield estimates of recurrence risk to siblings at a rate 10-60 times greater than the base rate of autism in the general population (reviewed by Santangelo and Tsatsanis, 2005).
Despite the severity and prevalence of this group of diseases, and with the likelihood that environmental influences are at least partially responsible, there have been no large scale epidemiological studies to date accessing geographic location, toxin exposure, and prevalence of autism (e.g. populations living near Superfund sites and prevalence of ASD).
One small case study by Edelson and Cantor (1998) recruited patients of private practice in Atlanta, Georgia, and employed glucaric acid analysis, high resolution gas chromatography coupled with electron capture detection (GC/ ECD) for blood analysis, and comprehensive liver detoxification evaluation. The authors found that 100% of the
20 autistic children studied (mean age= 6.4 years old) had abnormal liver detoxification profiles, and that 16 of 18 subjects (89%) with available blood analysis showed evidence 5 of levels of toxic chemicals exceeding the adult maximum tolerance. Some of the toxic chemicals included benzene, chloroform, and polychlorinated biphenyls.
Polychlorinated biphenyls (PCB) are different from many of the other toxins studied because they do not occur naturally. PCB compounds were manufactured as complex industrial mixtures from 1929 to 1977, mainly as nonflammable alternatives for capacitors, transformers, and plastics. PCB entered the air, water, and soil during their manufacture, use, disposal, and transport, as well as from leaks and fires of products containing PCB (ATSDR, 2000). Despite efforts to stop their production and use, these synthetic compounds are persistent, ubiquitous environmental threats because of their unique organic structure.
Figure 2: Structure of Polychlorinated Biphenyl (PCB) Molecule. Chlorine atoms can be placed on any combination of positions (para, meta, ortho), resulting in 209 congeners. www.epa.gov/hudson/images/pcb-mol.jpg
Polychlorinated biphenyls are formed by substitution of chlorine atoms on a dual-
ring structure comprised of two benzene rings linked by a single covalent carbon to
carbon bond between the 1 and 1’ positions (Figure 2) (http://www.epa.gov/hudson/pcbs
101.htm). With ten possible positions, at para (4 or 4’), meta (3 or 3’ and/or 5 or 5’), or 6 ortho (2 or 2’ and/or 6 or 6’) for chlorine substitution, there are 209 possible combinations, or congeners, of polychlorinated biphenyls. The number and positioning of the chlorine atoms determines the congener’s level of toxicity, based on toxic equivalencies to dioxin (Carpenters, 2006; Safe, 1994).
PCB congeners with no substitutions at the ortho-position are coplanar, whereas those with substitutions become increasingly noncoplanar (Lamb et al., 2006) because of steric hindrance around the covalent carbon-carbon bond. The lipophilic character, particularly of the more highly chlorinated congeners, chemical inertness, and thermal stability that made PCB of such valuable industrial use is what allows these aromatic hydrocarbons to bioaccumulate (Safe, 2004). The half-life of PCB compounds varies with congener and mixture, but one study of occupational exposure in workers showed the median half-life of Aroclor 1254 as 4.5 years (Phillips et al., 1989). PCB compounds collect in the adipose tissue of fish and marine mammals, reaching levels that may be many thousands of times greater than those in water. Thus, PCB exposure in mammals can be direct via consumption of contaminated food or water or indirect via perinatal exposure to maternal consumption through placental or lactational transfer (ATSDR,
2000).
Previous studies done in our lab have shown that dietary Aroclor 1254, a commercial mixture of PCB congeners, in either large amounts (62.5 ppm, 125 ppm, or
250 ppm) (Juárez de Ku et al., 1994) or smaller amounts (1.25 ppm, 12.5 ppm, or 25 ppm) (Provost et al., 1999) decreases circulating levels of thyroid hormones in a dose- dependent manner in rat offspring from PCB-fed dams. The importance of thyroid hormone in brain development has been extensively documented, making PCB-induced 7 neonatal hypothyroidism a possible useful model for understanding childhood developmental disorders like autism (Sadamatsu et al., 2006).
Whether directly through thyroid disruption or indirectly through other mechanisms, a vast amount of literature supports that PCB compounds can cause profound neuroendocrine disruption in rodents relevant to childhood developmental disorders. These include altered motor activity (Sugawara et al., 2005; Bowers et al.,
2004), learning (Lilienthal and Winneke, 1991; Corey et al., 1996), memory and attention
(Schantz et al., 1995), responsiveness to adverse stimuli (Daly et al., 1989), and sensory function (Crofton and Rice, 1999).
As reviewed by Nayate et al., (2005) behavorial assessment of infants with autism and Asperger’s syndrome have been found to display abnormal motor development, such as an abnormal pattern of righting, a lack of protective reflexes when falling, abnormal gait sequencing, delayed development through the stages of walking, and abnormal hand positioning. The cerebellum plays a critical role in the integrative control of locomotion.
Bauman and Kemper (2004) reported that the most apparent and consistent pathological abnormalities in autistic children have been confined to the cerebellum. To date, all of the brains they studied from autistic patients, regardless of age, sex, or cognitive abilities have shown a significant decrease in the number of Purkinje cells, primarily affecting the posterolateral neocerebellar cortex and adjacent archicerebellar cortex of the cerebellar hemispheres (Bauman and Kemper, 2004). Analagously, PCB-exposure has been implicated in disrupting cerebellar development (Nguon et al., 2005) and motor functioning (Sugwara et al., 2005; Bowers et al., 2004) in rodent models. Just as a child’s developmental milestones can be assessed with behavioral scales (e.g. NBAS), 8 behavioral paradigms can be used with rodents to assess activity and anxiety (e.g. open field activity), strength and coordination (e.g. hang test and negative geotaxis response), and developmental reflexes (e.g. righting response). It is believed that perinatal exposure to PCB toxins causes alteration in motor development, but the onset and time course of these deficits are unclear.
Neurological abnormalities have been reported in 30-50% of autistic patients.
These include but are not limited to hypotonia or hypertonia, disturbance of body schema, spasmodic movements, myoclonic jerking, drooling, abnormal posture and gait, and tremor (DeMeyer et al., 1973; Tsai, 1999). These behaviors are linked to dysfunction of the basal ganglia, specifically the neostriatum and its connections
(Rinehart et al., 2006). Several imaging studies have revealed structural abnormalities within the frontrostrial system in both individuals with autism and Asperger’s syndrome.
Sears et al., (1999) found increased caudate volume in individuals with autism, proportional to increased overall cortical volume, while Siegel et al., (1992) found reduced glucose metabolism in the left posterior putamen of individuals with autism.
These differences in cortical and caudate volume may reflect abnormal neuronal migration during prenatal development.
The study of species-specific action sequences in animal models provide an opportunity to study how neural systems like the neostriatum coordinate natural patterns of serial order (Cromwell and Berridge, 1996; Fentress, 1992). Damage to the neostriatum does not disrupt the ability to emit syntactic grooming chains; rather, the characterized four phases end prematurely (Cromwell and Berridge, 1996). This is because the location of grooming moves along the anterior-posterior axis throughout 9 development, beginning with paw strokes around the mouth and vibrissae as early as day
1-2. Grooming proceeds to phase II-III, around the eyes and ears by day 8, and finally to phase IV body licking by day 14-15 (Berridge, 1992; Colonnese, 1993).
As a model, the effect of PCB-exposure on rodent grooming is more behaviorally analogous to disturbance of body schema than to the usual increase of compulsive behavior seen in the autistic population. Thus, to encompass autistic individuals that maintain rigid habits, similar to individuals with obsessive-compulsive disorder (Tsai,
1999), rats can be trained to establish a habit and then be evaluated in a reversal task.
T-maze learning involves finding a food reward in one of two available locations at opposite ends of a T-shaped arena (Crawley, 2004). Rats exposed to PCB perinatally are expected to take a longer time to learn the routine and to be more resistant to change entering one location out of habit even when the food reward is removed (Schantz et al.,
1997).
Previous behavioral work in our lab focused on examining juvenile PCB-exposed rats in paradigms such as conditioned order preference (COP), play behavior, and social port interaction. Control rats show a preference for an odor when it has been associated with the mother, and social play behavior is the earliest form of nonmother-directed social behavior in the rat. Our findings indicated that PCB-exposed rats display a lesser ability to discriminate in the COP test (Cromwell et al., 2007, in press), display significantly less play behavior, and are significantly less socially investigative than controls (unpublished findings).
Similarly, many autistic children prefer to play by themselves, avoiding others, and show marked delays in forming bonds with family members (Hollander, 2003). As 10 reviewed by Nelson and Panksepp (1998), like normal children, infant rats display motivation to seek out maternal contact and care, and rats of many ages show evidence of specific social learning and individual recognition. Although the neurobiological nature of this attachment system is complex, one biomolecular pathway that clearly influences social behavior is the arginine-vasopressin (AVP) system (Wassink et al., 2004).
Arginine-vasopressin, also known as vasopressin or antidiuretic hormone, is a nonapeptide classically known to be synthesized in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) of the hypothalamus and transported through the axons of magnocellular neurons to be stored in the neurohypophysis (Figure 3)
(http://www.brainviews.com/abFiles/DrwHypopit.htm). The other mammalian nonapeptide neurohypophyseal hormone, oxytocin (OXY), is synthesized predominantly in the paraventricular nucleus (PVN) of the hypothalamus and differs from vasopressin by only two amino acid substitutions, isoleucine for phenylalanine at position 3; and leucine for arginine at position 8 (Hadley, 2000). Vasopressin can bind to oxytocin receptors, and expression of oxytocin and vasopressin genes appears linked (Insel et al.,
1999).
The genes encoding VP are in tandem array on chromosome 20 in humans, separated by 8 Kb of DNA. Each has 3 exons, and encodes a polypeptide precursor with a modular structure: an amino-terminal signal peptide, the vasopressin peptide, a carboxy- terminal glycopeptide, and a larger binding protein called neurophysin II. Vasopressin- neurophysin II (pressophysin) is a hormone- specific binding protein which forms a dimeric complex with vasopressin. Neurophysins and their associated hormones (in this 11 case, pressophysin and vasopressin) are derived from the posttranslational cleavage of a precursor protein, propressophysin.
Figure 3: The Neurohypophysis Projections of magnocellular hypothalamic axons to posterior pituitary (http://www.brainviews.com/abFiles/DrwHypopit.htm)
The neurophysin-hormone dimers are packaged in the neuronal soma in microscopically
visible granules (Landgraf and Neumann, 2002). They are delivered by intracellular
transport to the nerve terminals in the posterior pituitary. The granule contents are
released into the general circulation either from terminals which end directly on capillary
endothelial cells or on cells which adjoin the vessel wall (Hadley, 2000).
Two functionally distinct vasopressinergic systems can be defined based on
differences in the sites of action and release of AVP. The peripheral vasopressinergic
system encompasses the sites of action for AVP released into peripheral circulation:
vascular smooth muscle, liver, and kidney. Thus, peripherally circulating AVP is
responsible for the classic endocrine functions ascribed to this neurohormone such as
vasoconstriction, glycogen metabolism, and antidiuresis (Hadley, 2000). In the central 12 vasopressinergic system, on the other hand, AVP acts as a neuromodulator/ neurotransmitter regulating an array of CNS-mediated functions (Ring, 2005). Separate neuronal populations exist that synthesize vasopressin for central release, such as the parvocellular neurons of the PVN, the medial amygdala, and the bed nucleus of the stria terminalis (De Vries and Buijs, 1983).
Beginning with the pioneering work of de Wied in the 1970s (reviewed by
Landgraf and Neumann, 2004), early AVP behavioral studies were focused on its positive effects on avoidance learning and memory (Van Wimersma Greidanus, 1982). More recently, studies have demonstrated a role for AVP in neuroendocrine reactivity, social behaviors, circadian rhythmicity, thermoregulation, and autonomic function (Ring, 2005).
For example, vasopressin release from the amygdala seems to be involved in the generation of passive rather than active coping strategies in stressful situations (Wotjak et al., 1996).
Studies in monogamous voles have begun to elucidate the neuropeptidergic circuitry underlying the formation and maintenance of social bonding relevant to understanding social deficits in autistic disorders. Prairie voles (Microtus ochrogaster)
are monogamous, while their close relatives, Montane voles (Microtus montanus) are
polygamous as are most voles. Vasopressin is necessary for males to pair-bond with
females (Bielsky and Young, 2004). Ample numbers of vasopressin receptors occur in
the nucleus accumbens of the monogamous voles, but not in the polygamous species.
This nucleus plays a central role in mediating pleasure, and vasopressin activates this pathway. Gene therapy can be used to selectively overexpress vasopressin receptors in 13 the nucleus accumbens in the male rodent of a polygamous species, thereby shifting them to monogamous behavior (Young et al., 2005).
AVP modulates these emotional and prosocial behaviors, mainly via specific interactions with the AVP receptor V1a. There are three receptor subtypes for AVP
(V1a, V1b, and V2), but V1a is thought to play the dominant role in regulating behavior.
Accordingly, mice lacking the AVPR1a gene show profound behavioral anomalies that collectively resemble those of human autism, including impairment in social recognition and reduced anxiety-like behavior (Bielsky and Young, 2004). Initial investigations into the social behavior of Brattleboro rats, a naturally occurring vasopressin-deficient mutant, demonstrated a total disruption of social recognition (Van Wimersma Greidanus, 1982).
Social memory was restored in these animals by retrodialyzing vasopressin into the lateral septum (Engelmann and Landgraf, 1994). Another recent clinical study found that intranasally administered vasopressin enhanced human facial responses (as measured by electromyograms) to normally neutral social stimuli, suggesting that vasopressin might modulate the processing of social stimuli in humans similarly to what is observed in rodent models (Thompson et al., 2004).
When addressing the paucity of information on ASD, many suggest that the gap
lies in our lack of animal models, particularly those that evaluate perinatal effects that
could be impacting development (London and Etzel, 2000). Though several studies have
documented motor deficits as a result of perinatal PCB exposure (Hany et al., 1999;
Bowers et al., 2004; Sugwara et al., 2005), few have followed the development of a wide battery of skills and retested to see if these skills improve or resolve with age.
Specifically, no studies to date have evaluated the effects of PCB exposure on stereotypic 14 motor behavior like rodent grooming syntax. The studies discussed in this thesis are designed to provide further evidence that perinatal toxin exposure causes neuroendocrine disruption, and in particular, aim to show that the spectrum of deficits fall in the same motor and social domains affected in the ASD population. A general battery of motor tests, paradigms of stereotypic repetitive behavior, and measure of circulating vasopressin levels were assessed in the PCB-exposed developing rat. Two different PCB doses and three developmental time points were examined with the goal of inducing autistic-like symptoms for a potential animal model of ASD.
15
MATERIAL AND METHODS
2.1 Animals All procedures relating to the animals were approved by the Bowling Green State
University Institutional Animal Care and Use Committee (IACUC protocol #04-015).
Sprague-Dawley parents of rats used for this study were obtained from Harlan-Sprague-
Dawley (Indianapolis, IN, USA). The rats were kept in a temperature- and humidity-
controlled room (70° F ± 2 and 30-70%, respectively) with a 12-hour light-dark cycle
(lights on 0700, lights off 1900) throughout the studies. Female rats weighing 225-275g
were mated to males of the same strain. Once females were determined to be pregnant as
confirmed by a sperm positive vaginal smear, they were caged separately, and fed ad
libitum either standard rat chow for control groups or chow with PCB 47/77 added at 12.5
ppm or 25 ppm (w/w). PCB 47 (2,2’,4,4’-tetrachlorobiphenyl) and PCB 77 (3,3’4,4’-
tetrachlorobiphenyl) were obtained from Accustandard, Inc., New Haven, CT, USA.
Stock PCB was dissolved in absolute ethanol, mixed with 100 g of rat chow (Mowlan
Teklad, Madison, WI, USA), and the ethanol was allowed to evaporate. Equal amounts
of PCB 47- and PCB 77-containing diet were mixed together and formulation of 25 ppm and 12.5 ppm doses was done by adding the appropriate weight of this concentrated mixture to sufficient unaltered diet to give a weight of 1000 g, which was thoroughly mixed by prolonged tumbling of the sealed container. Control animals were continued on standard rat chow after conception.
Litters for T-maze testing and vasopressin EIA were standardized to 10 rats, 5
males and 5 females when possible. Half the litters used for motor testing were
standardized with all pups from initial litters tested. Preliminary findings from tests with
all pups did not differ significantly from data obtained from standardized litters. All pups 16 were housed in the maternal cage until weaning at PND 21, and then housed in groups of
2 or 5 by sex. For organizational purposes the first set of male and female pups were shared for tests of conditioned odor preference, social port, and play behavior at PND 13-
15 (Cromwell et al., 2007), while the second set of males were shared for a test of social recognition at PND 20-21 (Jolous-Jamshidi, 2007) (Table 1-2).
Table 1: Timeline for first set of litters tested.
5 Males and 5 Females randomly chosen from 14 litters or until significant changes observed. Shaded columns indicate tests for present studies.
Age PND 13-15 PND 14-16 PND 21 PND 28-32 PND 60-64 (Round 1) (Round 2) (Round 3) Test Conditioned Open Field, Weaned and Open Field, Open Field, odor preference, Hang, Pair housed Hang, Hang, social portb, Negative Negative Negative play behavior Geotaxis, Geotaxis, Geotaxis, Righting, Righting, Righting, Grooming Grooming Grooming
aCromwell et al., 2007 bunpublished findings
Table 2: Timeline for second set of litters tested. 5 Males from 14 standardized litters or until significant changes observed. Shaded columns indicate tests for present studies.
Age PND 20-21 PND 21 PND 25-29 PND 29
Test Social Weaned and all T-maze Decapitation for Recognition 5 males housed Learning and serum samples together Reversal for vasopressin detection cJolous-Jamshidi, 2007
17
2.2 Developmental Motor Skills
The following four behavioral measures were carried out at similar times of day
for two consecutive days, between the daylight hours of 8:00 am-5:00 pm, at three
different developmental ages: PND 14-16, PND 28-32, and PND 60-64 in male and female rats.
2.2.1 Open-Field Activity
The open-field test was performed for the first two rounds (Table 1) of
developmental testing in a 40 x 50 x 20 cm square open-field apparatus with a nine
square grid drawn on the floor. Each rat pup was transferred from the home cage directly
into the center of the open field and observed for 10 min. Locomotor activity was
recorded, and later scored for horizontal movement (crossing a line into a new square),
vertical rear counts (number of times the two front paws came off the ground), and rear
time (length of time spent with two front paws off the ground, including grooming). A larger open-field apparatus, 61 x 62 x 31 cm, was used for the adult rats at round 3, PND
60+.
2.2.2 Righting Reflex
Developmental righting reflex was tested by timing the number of seconds it took an animal placed on its back to turn over onto its four feet.
2.2.3 Hang Test
The hang test measured the animal’s grip strength by its ability to climb a 27 x 16
cm grid on a 30º incline within 60 sec. The animal was given a 0 if it fell off the grid,
100 if it reached the top within 60 sec, or a 60 if it hung on but did not reach the top 18 within 60 sec. A proportionately larger, 61 x 44 cm, grid at the same angle was used for the PND 60+ rats.
2.2.4 Negative Geotaxis
Negative geotaxis measured the animal’s ability to turn 180° when placed head facing downward on a 30º incline 27 x 16 cm grid. The animal was scored 100 if they were able to turn around or a 0 if they could not. A proportionately larger, 61 x 44 cm, grid at the same angle was used for the PND 60+ rats.
2.3 Stereotypic Repetitive Behavior:
Grooming behavior was tested by an observer blind to pup treatment at similar
times of day for two consecutive days between the hours of 8:00 am-5:00 pm at three
different developmental ages: PND 14-16, PND 28-32, and PND 60-64 in both male and
female rats. T-maze learning acquisition and reversal learning was tested in developing
25-29 day old male rats during the daylight hours 8:00 am-12:00 pm.
2.3.1 Grooming Behavioral Testing:
Grooming sequences were recorded during 10 min test sessions in a 30.5 x 30.5 x
30.5 cm plexiglass front box. Later, a mirror-system was added beneath to allow
observation at more angles. Grooming was elicited by lightly spraying the dorsal side of the torso of the rat with a water mist. The rat was allowed to habituate for 5 min to the test environment before being sprayed. All videotaped grooming sequences were scored as described below by an observer blind to pup treatment.
19
2.3.1.2 Grooming Syntax Analysis
The serial organization of syntactic grooming chains arranges into four consecutive sequential phases as follows (Figure 4). Phase I: A concatenation of five to nine small, rapid bilateral forepaw strokes (“rapid ellipses”) around the nose and mouth at a rate of 6-7 Hz. Ellipse stroke movements at this speed are extremely rare outside of syntactic chains. The concatenation of multiple ellipse strokes, faster than 6 Hz, virtually never occurs during nonchain grooming. A fast series of Phase I ellipse strokes serves as the marker for the initiation of syntactic chains. Phase II: A short bout of one to four
Figure 4: Choreographed ``Syntactic Chain'' Sequence of Grooming Actions. A choreographic transcription of a prototypical syntactic grooming chain shows the moment-by-moment trajectories of forelimb strokes over the face and the occurrence of other grooming actions. Drawings of the rat display the actions that typify each phase of syntactic chains. The horizontal axis represents the position of the rat's nose, and stroke trajectories over the face are depicted relative to the nose. Deviations of the lines above (right paw) and below (left paw) the horizontal axis represent the elevation (level of the eye, the ear, etc.) reached by each forepaw during a stroke. Small rectangles denote paw licks. Large rectangle denotes body licking. (Cromwell and Berridge, 1996)
20 small or medium sized paw strokes along the mystacial vibrissae, usually performed by one unilateral paw or by both paws tracing asymmetric amplitudes. Phase III: A repetitive series of 3-10 large bilaterally symmetrical strokes, which may extend behind the ears and most of the head. Phase IV: A bout of body licking over the lateral and ventral torso
(Figure 4). Once the initial components of Phase I appear, the entire sequence follows to
Phase IV with a completion rate of 85-95% for normal adult rats (Cromwell and
Berridge, 1996).
Grooming behavior was analyzed for efficacy of syntactic completion following the Cromwell and Berridge (1996) scoring protocol. Once initiated, grooming chains were analyzed for syntactic completion rates for each group to assess the ability to implement full sequential patterns. A “syntactically perfect” complete chain was defined as one that progressed through Phases I, II, III, and IV (body licking, in which the rat lowered its head after the last Phase III stroke and turned sideways in order to bring its tongue in contact with its flank or back), without interruption and within 5 sec of Phase I.
“Incomplete” chains were considered to be those in which the rat reverted to sequentially flexible grooming within the chain or in which the rat simply stopped grooming before
Phase IV. “Complete Imperfect” chains were those that progressed through Phases I, II,
III, and IV, but did not show the typical body licking. For example, imperfect chains may have ended in paw or shoulder licking.
A grooming “bout” was defined as non-chain grooming, thus “total grooming” included both grooming bouts and chain grooming. “Total chains initiated” is the sum of attempts to elicit a grooming chain, as indicated by Phase I, within the two ten minute testing sessions. 21
2.32 T-maze Acquisition and Reversal
The effort-based decision-making t-maze task, (adapted from Crawley, 2004;
Moy et al., 2006) consisted of an approach arm and two goal arms (77 x 135 x 14 cm).
Animals were first given a 25 mg sucrose pellet in the home cage, habituated to the
t-maze arena for 3-5 mins, and then food deprived for 12-14 hr before day one of testing.
At the beginning of each test session, the rat was placed in the start box at the bottom of
the approach arm. The start box door was then opened, and the rat was given a choice of
entering either arm. In this manner, the rats were trained, with 10 trials on two days of testing, to establish a habit of finding a food reward at the assigned goal arm. The reinforced arm was randomly assigned. A correct attempt was defined as when the rat entered the reinforced arm and consumed the sucrose pellet, while an incorrect attempt was defined as when the rat crossed the marked line (40 cm into the arm) in the non-goal arm. If the rat made a correct choice, it was confined to the correct arm by closing a door, and given time to consume the pellet before being guided back to the start box. If the rat made an incorrect choice, the door was closed as soon as the forepaws crossed the marked line, and the rat was confined to the incorrect side with no reinforcement for five seconds before being guided back to the start box.
After a minimum of two days of training, those rats meeting 80% criterion, 8/10
trials correct, were tested with the reversal task. The food reward was changed to the arm
opposite that used in learning acquisition, and the number of correct choices of 10 trials
was determined for each rat. Those rats who did not meet 80% criterion within the 5
days of testing were not used in the reversal task.
22
2.4 Vasopressin Enzyme Immunoassay
Circulating vasopressin concentrations were measured using an enzyme
immunoassay (EIA kit, Assay Designs, Ann Arbor, USA) with sensitivity of 3.39 pg/ml
of analysate. Blood from decapitation of male rats at PND 29 was collected directly into
cryovials and centrifuged at 2000 x g for 15 min after blood collection. Decapitation was
carried out consistently at peak daylight hours, 1:30-4:30pm, when the neurohypophyseal
hormones are the most detectable in rodents (Forsling, 2000).
Because the vasopressin content in normal serum is very low, serum samples
were first extracted using 200 ml C18 Sep-Pak columns and then concentrated using a centrifugal concentrator (Speedvac Concentrator, Savant Instruments Inc, Farmingdale,
NY) according to the EIA manufacture’s instructions. Samples were incubated in goat
anti-rabbit IgG antibody-coated microtiter wells with rabbit anti-AVP polyclonal
antibody and alkaline phosphatase-congugated AVP for 22 hrs at 4° C, after which the
liquid contents were emptied and the wells incubated with p-nitrophenyl phosphate for 1
hr at room temperature. The enzyme reaction was stopped with a solution of trisodium
phosphate in water and the absorbance values of the yellow color generated were read 1
sec per well on a microplate reader (Victor2 1420 Multilabel Counter, Wallac
Laboratories, Turku, Findland), set at 405 nm.
Duplicate readings were averaged for standards, controls, and samples for each
assay. A standard curve was generated (Microsoft Office Excel, 2002) using the
calculated percent bound (y-axis) against the known concentrations of vasopressin for the
standards (x-axis). A best fit line was drawn through the data points and the 23 corresponding best fit line equation was used to determine the vasopressin concentrations.
2.5 Statistical Analysis:
Data for the hang, negative geotaxis, and righting reflex were ranked and
subsequently analyzed using a general linear model of nonparametric analysis of variance
(NPAR1WAY-GLM), allowing comparisons of differences between means of male and
female groups among treatment groups. Grooming measures from day 1 and day 2 were
combined and then analyzed using nonparametric Kruskal-Wallis analysis of variance
(ANOVA) tests for each developmental time point (SAS v. 9.0, SAS Institute Inc., Cary,
NC).
Comparison of mean open field data was analyzed with an analysis of variance
(ANOVA). T-maze learning was analyzed using nonparametric Kruskal-Wallis ANOVA
and t-maze learning competency was assessed using Chi-square. T-maze reversal tasks
and circulating vasopressin were analyzed using one-way ANOVA (SPSS 13.0). For all
comparisons, significance was set at p<0.05. If a significant main or interaction effect
was detected, either a nonparametric Mann-Whitney U or parametric Tukey post hoc
analysis was performed (p=0.05) for pairwise means comparisons. 24
RESULTS
Developmental Motor Activity:
Open Field Activity: Comparisons of mean activity levels showed that rats exposed to PCB demonstrated a significant decrease in horizontal movement as adults (p<0.0001)
(Figure 5), and a significant increase in both mean rear counts (p<0.05) (Figure 6) and mean time spent rearing (p<0.05) as adolescents (Figure 7, Table 11). There were no significant differences at the juvenile age for any open field parameters tested.
110
100
90
80 *
70 *
60 Mean HorizontalMean Crosses
50
40 0123 Round
Control 12.5 ppm 25 ppm
Figure 5: Effect of PCB on Developmental Mean Horizontal Movement. Horizontal crosses = number of times the animal crossed a line into a new square in a 10 minute open field test session. Developmental Time: Round 1=PND 14-16, Round 2=PND 28-32, Round 3=PND 60-64. *Significantly different from control (p<0.05)
25
60
50 * 40 *
30
20 Mean Rear Counts Mean
10
0 0123 Round
Control 12.5 ppm 25 ppm
Figure 6: Effect of PCB on Developmental Mean Rearing. Rear count= number of times two front paws came off the ground in a 10 minute open field test session. Developmental Time: Round 1=PND 14-16, Round 2=PND 28-32, Round 3=PND 60-64. *Significantly different from control (p<0.05)
120
100 * 80 *
60
40 Mean Rear Mean (sec)Time
20
0 0123 Round
Control 12.5 ppm 25 ppm
Figure 7: Effect of PCB on Developmental Mean Rear Time. Rear time= length of time (sec) the animal is off its front paws (includes grooming). Developmental Time: Round 1=PND 14-16, Round 2=PND 28-32, Round 3=PND 60-64. *Significantly different from control (p<0.05) 26
Table 3: Sample Size for all Open Field Parameters Tested. Condition PND 14-16 PND 28-32 PND 60-64
Control n= 16 n= 12 n= 16 12.5 ppm n= 24 n= 28 n= 16 25 ppm n= 22 n= 16 n= 16
Hang Test: Significant differences in mean hang score were determined using the general linear model, [y= Cond + Sex + (Cond x Sex)]. Control rats performed better at all time
points tested, with hang score improving throughout development. PCB 12.5 rats were
most affected as young pups, testing significantly 33% lower (p<0.01) on hang
performance in comparison to controls, while the greater dose PCB 25 rats were most
affected at adolescence, a 23% significant depression (p<0.05) of hang performance in
comparison to controls. Both PCB 12.5 and 25 groups were significantly affected in
adulthood (p<0.001), 42% and 44% deficient in hang performance relative to controls,
respectively (Figure 8, Table 11). Pairwise comparisons of males and females showed no significant differences. 27
100
90
80
70
60
Hang ScoreHang (0-100) * 50 * * * 40
30 0123 Developmental Time (Round)
Control 12.5 ppm 25 ppm
Figure 8: Effect of PCB on Developmental Mean Hang Test. Hang Score= 100 if reach top of incline grid, 0 if fall off, 60 hang on but do not reach the top, within 60 sec. Developmental Time: Round 1=PND 14-16, Round 2=PND 28-32, Round 3=PND 60-64. *Significantly different from control (p<0.05)
Table 4: Sample Size for Mean Hang Test: Condition PND 14-16 PND 28-32 PND 60-64
Control n= 26 n= 28 n= 12 12.5 ppm n= 40 n= 32 n= 32 25 ppm n= 38 n= 36 n= 22
Negative Geotaxis Response: Similar to hang ability, control rats consistently scored better, with negative
geotaxis response improving linearly throughout development (Figure 9). Pairwise
comparison of the interaction effect of treatment and sex within developmental time
revealed significant deficits in negative geotaxis response in adolescence (p<0.0001), with performance scores 40% less in 12.5 ppm and 48% less in 25 ppm in comparison to 28 controls (Figure 9, Table 11). These deficits in negative geotaxis ability improved nearly
20% when pups were retested at round 3, resulting in no significant differences between groups in adulthood. Further analysis showed a significant overall interaction between sex and treatment (p<0.05). Round 2 males were significantly affected by PCB 25 ppm
(p<0.05), while females were significantly affected by both PCB 12.5 ppm (p<0.0001) and PCB 25 ppm (p<0.01). There were no significant differences between sexes at round
3.
100
90
80
70
60
50 *
Negative (0-100) Geotaxis Score 40 *
30 0123 Developmental Time (Round)
Control 12.5 ppm 25 ppm
Figure 9: Effect of PCB on Developmental Mean Negative Geotaxis Response. Negative Geotaxis Score= 100 if turn 180° when head down on incline grid, 0 if no turn observed or fall off. Developmental Time: Round 1=PND 14-16, Round 2=PND 28-32, Round 3=PND 60-64. *Significantly different from control (p<0.05)
Table 5: Sample Size for Mean Negative Geotaxis Response.
Condition PND 14-16 PND 28-32 PND 60-64
Control n= 20 n= 28 n= 10 12.5 ppm n= 40 n= 32 n= 32 25 ppm n= 32 n= 36 n= 22
29
Righting Reflex: Control rats were able to right themselves at all time points tested, while PCB 25 rats showed a significant righting latency at round 1 of testing (p<0.01) (Figure 10, Table
11). This deficit was later overcome with no differences observed between control and treatment groups when righting was retested at round 2 and round 3. Pairwise comparisons of males and females showed no significant differences.
1.16
1.14
1.12
1.10 *
1.08
1.06
1.04 Righting (seconds)
1.02
1.00
0.98 0123 Developmental Time (Round)
Controls 12.5 ppm 25 ppm
Figure 10: Effect of PCB on Developmental Mean Righting Reflex. Righting= length of time (sec) the animal takes to right itself on all four paws when placed on its back. Developmental Time: Round 1=PND 14-16, Round 2=PND 28-32, Round 3=PND 60-64. *Significantly different from control (p<0.05)
Table 6: Sample Size for Mean Righting Reflex
Condition PND 14-16 PND 28-32 PND 60-64
Control n= 26 n= 26 n= 10 12.5 ppm n= 32 n= 32 n= 32 25 ppm n= 30 n= 36 n= 22 30
Stereotypic Repetitive Behavior:
Grooming: Despite similar rates of total grooming (Figure 11) and chains initiated (Figure
12) for all control and treatment groups, rats exposed to PCB 25 displayed a significant delay in the formation of syntactic chain grooming (p<0.0001) (Figure 13). At round 1,
PCB 25 rat pups showed a 0% completion rate in comparison to the 25.9% completion rate exhibited by controls and PCB 12.5 pups of the same age (Figure 13, Table 11).
PCB treated rats showed some deficit in chain completion into adulthood, but these differences were not significant (Figure 13). Moreover, PCB 25 rat pups showed a significantly higher percent of incomplete chains at round 1 in comparison to controls
(p<0.0001) (Figure 14). There were no significant differences observed between control and treatment groups for CI grooming at all ages tested.
31
4.0
3.5
3.0
2.5
2.0 Mean Total Grooming/Rat Total Mean
1.5
1.0 0123 Developmental Time (Round)
Control 12.5 ppm 25 ppm
Figure 11: Mean Total Grooming/ Rat. Mean total grooming per rat is the average number of flexible bouts + chains per grooming test session. Developmental Time: Round 1=PND 14-16, Round 2=PND 28-32, Round 3=PND 60-64.
4.0
3.5
3.0
2.5
2.0 Mean Chains Initiated/Rat Chains Mean
1.5
1.0 0123 Developmental Time (Round)
Control 12.5 ppm 25 ppm Figure 12: Mean Chains Initiated/ Rat. Mean chains initiated per rat is the average number of Phase I grooming counted per grooming test sessions regardless of syntactic completion. Developmental Time: Round 1=PND 14-16, Round 2=PND 28-32, Round 3=PND 60-64. 32
100
80
60
40
20 % Complete Perfect% Chains 0 * 0123 Developmental Time (Round)
Control 12.5 ppm 25 ppm
Figure 13: Percent Complete Perfect Chains. CP chains are defined as following the characterized four phases from initiation to completion. Developmental Time: Round 1=PND 14-16, Round 2=PND 28-32, Round 3=PND 60-64. *Significantly different from control (p<0.05)
100 * 80
60
40
20 % Incomplete Chains %
0
0123 Developmental Time (Round)
Control 12.5 ppm 25 ppm
Figure 14: Percent Incomplete Chains. Incomplete chains are defined as chains that lack Phase IV grooming. Developmental Time: Round 1=PND 14-16, Round 2=PND 28- 32, Round 3=PND 60-64. *Significantly different from control (p<0.05) 33
12
10
8
6
4
2 % Complete Chains Imperfect Complete % 0
0123 Round (Developmental Time)
Control 12.5 ppm 25 ppm
Figure 15: Percent Complete Imperfect Chains. CI chains are defined as complete chains with atypical Phase IV grooming, such as paw licking rather than dorsal body licking. Developmental Time: Round 1=PND 14-16, Round 2=PND 28-32, Round 3=PND 60-64.
Table 7: Sample Sizes for all Grooming Parameters Tested. Condition PND 14-16 PND 28-32 PND 60-64
Control n= 24 n= 23 n= 15 12.5 ppm n= 32 n= 32 n= 35 25 ppm n= 30 n= 34 n= 29
34
T-maze: PCB exposure caused a significant latency in t-maze learning acquisition in a dose dependent manner (p<0.0001) (Figure 16, Table 4).
4 *
3 *
2 # Learning Days
1
0 0 ppm 12.5 ppm 25 ppm Treatment Group
Figure 16: Mean Learning Days Needed to Establish a Habit: Effect of PCB on T-maze Acquisition Learning in Juvenile male rats. Mean learning days needed to meet criterion of 80%, 8/10 trials correct. *Significantly different from control (p<0.05)
Table 8: Sample Size for Mean Learning Days.
Treatment Group # Learning Days Required
Control n=26 12.5 ppm n=18 25 ppm n=25
35
Significantly fewer PCB 25 animals were unable to meet the 80% criterion to move onto the reversal task in comparison to the 12.5 and control groups (p<0.01) (Table 9).
Correspondingly, PCB exposed rats showed significantly less competency in the reversal task in a dose dependent manner (p<0.0001) (Figure 17).
Table 9: % Meeting Competency Criteria Percent of animals meeting criterion (8/10 correct trials in one test session) within the 5 days of testing. Treatment Group % Meeting Competency Criteria
Control 100 n=26 12.5 100 n=18
25 76 a n=25 Values represent mean % meeting competency ± standard error of the mean a Significantly different from control group (p<0.05)
36
100
80 *
60 *
40
20 Reversal Score (% ReversalCorrect/10) Score (%
0 0 ppm 12.5 ppm 25 ppm Treatment Group Figure 17: Accessing Adaptability: Effect of PCB on Mean T-maze Reversal Learning in Juvenile male rats. Reversal Score= number of correct choices out of 10 when food reward moved to opposite goal arm. *Significantly different from control (p<0.05)
Table 10: Sample Size for T-maze Reversal. Treatment Group % Correct Reversal Control n=26
12.5 ppm n=18
25 ppm n=19 37
Table 11: Summary of Behavioral Results in Rats Exposed to PCB 47/77 at 0 ppm, 12.5 ppm and 25 ppm. ↓ = significant decrease relative to controls, ↑ = significant increase relative to controls, Ø = no significant difference from controls, Horz= mean horizontal open field activity, Rear # = mean number of rears, Rear time = mean length of time spent rearing and grooming, ND = not done
Juvenile Adolescent Adult
Open Field Activity Ø ↑ 12.5/25 Rear # and ↓ 12.5/25 Horz time
Hang Test ↓ 12.5 ↓ 25 ↓ 12.5/ 25
Negative Geotaxis Ø ↓ 12.5/ 25 Ø
Righting Reflex ↓ 25 Ø Ø
Grooming Syntax ↓ 25 Ø Ø
T-maze Reversal ND ↓ 12.5/ 25 ND
38
Neuroendocrine Status:
Serum Vasopressin Concentrations: No significant differences were found in mean circulating vasopressin concentrations (Figure 18).
10
8
6
4
2
Circulating Vasopressin Concentrations (pg/ml) Concentrations Vasopressin Circulating 0 Control 12.5 ppm 25 ppm Treatment Group
Figure 18: Effect of PCB 47/77 on Mean Circulating Serum Vasopressin Concentrations in 29-Day Old Male Rats.
Table 12: Sample Size for Serum Vasopressin Analysis. Treatment Group Vasopressin Concentration (pg/ml) Control n=26 12.5 ppm n=18 25 ppm n=25
39
DISCUSSION General Motor Activity In the present study, a general battery of motor tests observed deficits that
appeared at various times in development, with some deficits becoming more pronounced
with age, while others improved and eventually resolved (Table 11). These results
support several other studies that have reported PCB disruption to general activity and
motor skills. Since factors such as PCB congener, dose, duration and type of exposure,
species, and age at assessment can confound comparison of PCB effects on overall locomotor activity levels; a comparison among studies has been included to aid in data interpretation (Table 13).
Similar to the juvenile rats in the present study, Bowers and colleagues (2004)
found that a PCB mixture, Aroclor 1254, delayed the righting reflex and reduced grip strength (equivalent to the present hang test) in rat pups at PND 10-14. Interestingly, the
present study found PCB 25 to delay righting reflex (Figure 10), but only PCB 12.5 to
decrease hang ability (Figure 8) in the rat pups tested. This difference is most likely due
to the higher dose (15 mg/kg/b.wt.) of complex PCB mixture (Aroclor 1254) given by
Bowers et al., (2004) rather than the present study’s simple two congener mixture at
lower doses (PCB 47/77 at 1.04 and 2.08 mg/kg/b.wt.) (Table 13).
Sugawara et al., (2005) observed decreased walking speed in the open field in
adult, PND 60, PCB-exposed mice relative to control mice. The present study also
observed decreased walking speed in similar aged adult rats as indicated by significantly
fewer mean horizontal crosses over a constant time (Figure 5).
There has been conflicting evidence and debate as to whether PCB toxins cause
hypo- or hyperactivity. Though this is certainly a function of PCB congener, dose, and 40
Table 13: Comparison of PCB Congeners/ Doses and Motor Outcomes. Doses have been standardized to mg/kg/ b.wt. for comparison to the present studies. Neg Geo= mean negative geotaxis reflex, Right= mean righting reflex, Subcu= subcutaneous injection. Symbols have been included to indicate specific dose outcome. Ø =no significant difference with all doses tested, € =significant difference for all doses tested.
PCB Daily Species Juvenile Adolescent Adult Congener/ Maternal Exposure PCB Dose mg/kg/b.wt
ℓ † Current PCB 47/77 1.04 , 2.08 Sprague- Ø Open Field ↑ Open Field€ ↓Open Field€ Studies Dawley GD 1- PND Rat ℓ € ↓ Hang Hang† ↓ Hang 21 ↓
in rat chow Ø Neg Geo Ø Neg Geo Neg Geo€ ↓ ↓ Right† Ø Right Ø Right Bowers et Aroclor 1254 15 Sprague- ↓Hang € al.,2003 Dawley GD 1- Rat ↓Right € PND 23 on cracker Ω Sugawara Aroclor 1254 0, 6, 18 , 54 C57BL/ ↓ Neg Geo€ ↓ Open FieldΩ et al., 2005 6Cr GD 6- Mouse PND 20 gavage Hany et PCB 77 0.5 (PCB77)₤ Long- Ø Open Ø Open Field al., 1999 Evans Field₤∞ PCB 47/77 1.5 (PCB 77)∞ Rat
GD 7-18 1/ 0.5 (PCB Subcu 47/77)
Nguon et Aroclor 10 Sprague- ↓ NegGeo€ al., 2005 1254 Dawley Rat ↓ Right€ GD 11- PND21 gavage
Bushnell et Aroclor 1254 6 Long- Ø Open Field al., 2002 Evans GD 6- Rat PND 20 gavage 41 mode of administration, the present data show that age tested is an imperative factor.
Few studies have examined an intermediate age such as PND 28-32, and the present study found that rats exposed to PCB were hyperactive at this age in comparison to like- age controls. Significant changes in adolescent activity were observed with an increase in mean rear counts (Figure 6) and mean time spent rearing and grooming (Figure 7).
Similar to previous findings (Bushnell et al., 2002; Hany et al., 1999), there were no significant differences in open field activity at the juvenile age tested (Table 11).
Specifically relevant to the disproportionate number of boys with ASD, a recent study by Nguon et al., (2005) found that cerebellar mass was more greatly suppressed in male rodents perinatally exposed to PCB than PCB-exposed females. Differential changes in the behavior such as righting reflex and negative geotaxis of PCB pups were associated with alterations in cerebellar structure and protein expression, with greater effects in males. Because cerebellar development coincides with the period of maturation of the hypophyseal-pituitary axis, it is tempting to suggest that the sex-specific nature of the response to PCB compounds is induced by alterations of thyroid hormone. Though significant depression in righting (juvenile-aged) and negative geotaxis response
(adolescent-aged) were seen in the present study, there were few if any sex differences observed. This difference is most likely attributed to the much higher dose (10 mg/kg/b.wt.) and more complex mixture (Aroclor 1254) administered by the latter study
(Nguon et al., 2005) in comparison to the present study (PCB 47/77 at 1.04 and 2.04 mg/kg/b.wt.). The present study found males were more significantly affected by high dose PCB 25, while females were significantly affected by both PCB 12.5 and PCB 25 42 ppm in hang ability tested at round 2. It would be necessary to test more animals and to further investigate a potential sex difference.
Overall, the motor deficits displayed by the PCB-exposed rats in the present study are similar to several abnormalities reported in infants and children with autism and
Asperger’s syndrome (Nayate et al., 2005): abnormal pattern of righting (righting reflex latency, Figure 10), a lack of protective reflexes when falling (negative geotaxis, Figure 9 and hang ability, Figure 8), and changes in activity levels (open field horizontal crosses, rear counts, and rear time, Figures 5-7). In particular, the later onset of symptoms seen with rats exposed to PCB 12.5 ppm in comparison to PCB 25 ppm mimics the differences in symptom onset seen between individuals with autism and Asperger’s syndrome.
Though PCB accumulation was not measured in the present rat pups tested, a study by Hany et al., (1999) used the same combination of congeners and similar doses as the present study (Table 14), and the authors found that PCB 47 accumulated to a 15-
30 fold greater degree in dams at GD 19 and at weaning (PND 21) and in the offspring at
GD 19 but not at weaning, when a >150-fold accumulation occured. This was attributed to a greater lactational transfer along with a slower metabolic degradation of PCB 47 than
PCB 77. Biotransformation selects for hydroxylation of the meta and para positions, yielding elevated proportions of non-coplanar ortho-substituted congeners (Fischer et al.,
1998). Thus, the mobilization of body fat during lactation could result in relatively higher PCB 47 concentrations compared to PCB 77. Since the present study observed motor skills before and after weaning, the ratio of PCB 47/77 would have fluctuated, potentially explaining some of the behavioral variation observed. 43
Both PCB 47 and PCB 77 were chosen for our investigations because they are structurally similar to thyroid hormones, with the same degree of halogenation.
Additionally, both congeners represent archetypes of different modes of action, and, at the same time, they possess structural comparability. PCB 77 and other coplanar dioxin- related toxins can impact the developing brain at the genome through the arylhydrocarbon receptor, AhR. When the toxin binds to the ligand-activated receptor,
AhR dissociates from its tetrameric complex and translocates into the nucleus, forming a heterodimer with the AhR nuclear translocator molecule (ARNT) (Shimba et al., 2001).
This heterodimer binds to dioxin responsive elements (DRE) in the DNA and regulates transcription of target genes, including xenobiotic-metabolizing enzymes (Shimba et al.,
2001). In contrast, non-coplanar ortho-substituted congeners such as PCB 47 induce
CYP2B1/CYP2B2 genes through AhR-independent mechanisms. PCB 47 has recently been shown to be more neurotoxic than its coplanar counterparts (Tilson and Kodavanti,
1998). The behavioral alterations seen in the present study are consistent with the hypothesis that developmental neurotoxicity of PCB compounds is due, in part, to the presence of ortho-substituted PCB congeners.
While the exact mechanism(s) of the effects of PCB in the current study remain unknown, these data suggest that PCB exposure at even low doses has the ability to cause profound motor disruption. Thus, PCB exposure is a potential environmental factor capable of interacting with genetic factors to cause a wide range of dynamic behavioral deficits.
44
Stereotypic Behavior
In the present study, PCB 25 rat pups showed a significant delay in the formation of syntactic chain grooming in comparison to controls of the same age (Figure 13).
While the individual behaviors composing chain grooming are likely generated in the hindbrain (Berridge, 1989), the sequential coordination of grooming syntax depends on dopamine (DA) neurotransmission in basal ganglia circuits (Matell et al., 2006). PCB exposure has been shown to cause significant dose dependent reduction in DA levels in developing rat striatum and ventral mesencephalon (VM) co-cultures (Lyng et al., 2007).
Lyng and colleagues (2007) reported increased neuronal cell death in both the VM and striatum and reduced numbers of DA neurons in the VM. While the mechanism of DA reduction with PCB exposure is unclear, in vitro PCB experiments have reported reduction of tyrosine hydroxylase levels (Lyng et al., 2007), the rate limiting enzyme
involved in dopamine biosynthesis, by reduction in dopamine transporter (DAT)
expression (Lyng et al., 2007; Caudle et al., 2006). PCB-induced inhibition of DA reuptake can result in the accumulation of unsequestered DA metabolites, resulting in oxidative stress (Fahn and Cohen, 1992; Lee and Opanashuk, 2004), cell injury, and eventually cell death (Lee and Opanashuk, 2004).
Though it is likely that the present results reflect a developmental delay of chain
syntax at the level of the basal ganglia and dopamine circuitry, it is possible that PCB
exposure is altering maternal care. Maternal contributions such as pup retrieval, nursing,
licking, and grooming are important for offspring development. It is believed that PCB
exposure increases (Simmons et al., 2005) maternal care and grooming, but whether this
causes an overall increase or decrease in grooming received is unclear. Perhaps when 45 maternal care changes, there could be compensation with increased or decreased self grooming or allogrooming. Nonetheless, alteration of maternal care could delay the development of complete chain syntax that usually appears on PND 14 at a typical 20% completion rate (Colonnese, 1993). Future studies using the same doses of PCB 47/77 might investigate this further by not only measuring self-directed grooming and maternal licking and grooming, but also effects of cross-fostering on grooming, as well as differences in grooming frequency by or on males versus females, respectively.
Another possible explanation of the inability of the PCB 25-exposed pups to complete chains may be centered in the disruption of the rat pup’s balance. During experimentation, it was observed that when control rats successfully completed a chain, they were oftentimes leaning against the corner of the testing chamber. The PCB 25 rat pups were able to initiate grooming, but abruptly stopped chain syntax by Phase II or III and usually fell upon doing so. As previously mentioned, there is a vast amount of literature, including the general battery of locomotor results discussed here, that suggest
PCB compounds disrupt the cerebellum. In addition, a recent study by Crofton et al.,
(2000) showed that perinatal PCB administration in rats (8 mg/kg) causes the loss of low- frequency hearing via loss of outer hair cells in the organ of Corti. Damage to the cochlea could then result in vestibular changes causing imbalance. Therefore, future studies should stabilize the rat pups with a support string (Colonnese et al., 1996) to distinguish if it is instability or disruption to the neostriatum that is causing the developmental delay.
Dopaminergic mechanisms have also been shown to play a role in modulating learned behaviors, particularly appetitive instrumental learning. For instance, dopamine 46 has been proposed to encode deviations from reward expectation (Schultz and Dickinson,
2000), reflect the incentive salience of reward prediciting cues (Berridge, 2001), and promote the transition from cognitively mediated to automatic habitual behavior (Everitt et al., 2001). In the present study, PCB exposed 25-29 day old male rats tested in the t- maze were significantly delayed in acquisition learning (Figure 16) and significantly less able to reverse the task (Figure 17) in a dose dependent manner in comparison to control rats. 24% fewer PCB 25 rats met the 80% criterion to move on to the reversal task in comparison to the 100% competency of the 12.5 treatment and control groups (Table 4), suggesting the PCB 25 rats were having difficulty learning the location of the food reward. Then, once having learned that location, the PCB exposed rats had difficulty adapting to a new food location.
Several recent sets of data have suggested that a broadly distributed network of striatal, cortical, and limbic sites subserve this instrumental learning process (reviewed in
Kelley, 2004). It is thought that the nucleus accumbens (NAc), a brain region located within the ventral striatum, is the interface between motivation and action. The NAc integrates information related to cognitive and emotional processing and its interactions with the prefrontal cortex and the amygdala work to control appetitive learning. Despite strong reciprocal connections, the ABL has been shown to be proportionately more involved in mediating the acquisition of incentive value by cues (habit formation), while the OFC is more involved in using these representations to subsequently guide responses necessary for reversal tasks (adaptation) (Schoenbaum et al., 2000). The present t-maze
PCB dose dependent deficits warrant further investigation into the cortico-limbic-striatal circuitry that is potentially being disrupted (Schoenbaum et al., 2003). Additionally, as 47 expected, these data are consistent in modeling both the challenge of learning tasks
(particularly those that require sensory input) and the rigid habit formation characteristic of the ASD population (Hollander, 2000).
Endocrine Status: Vasopressin
Enzyme immunoassay analysis revealed no difference in circulating vasopressin
concentrations between PCB-exposed animals and control animals (Figure 18). There
have been no previous studies to date assessing the effect of PCB on emotionally stimulated (CNS) AVP release, but there have been a few studies evaluating the effect of
PCB compounds on osmotically stimulated (PNS) AVP release. A study by Coburn et al.,
(2004), explored the effect of Aroclor 1254 on central and peripheral AVP release in
response to dehydration in adult rats. They found that PCB-exposed rat brains,
specifically the supraoptic nucleus (SON), failed to respond with increased AVP release
during dehydration. Further investigation into structure-activity relationships found PCB
47 was neuroactive, while PCB 77 did not significantly reduce AVP release (Coburn et
al., 2007). While the present study measured AVP in younger 29-day old rats, with
potentially less AVP and more PCB 47 (Hany et al., 1999), this would not explain the
neurobehavioral changes seen in our environmentally stressed animals.
As previously discussed, AVP has been traditionally understood as being
synthesized on ribosomes in the soma of neurons in the SON, post-translationally
processed in neurosecretory vesicles in the axon, and stored for systemic release at the axon terminals in the posterior pituitary (Hadley, 2000). A recent study by Ma and
Morris (2002) confirmed that magnocellular hypothalamic neurons are capable of 48 synthesizing proteins within their dendrites. This suggests that separate and possibly independent neuropeptide synthesis in the somata (for peripheral secretion and somatic release) and in the dendrites (for dendritic release within the brain) contributes to independent release patterns, making measurements of AVP in serum an unreliable guide to AVP changes within the brain. As reviewed by Landgraf and Neumann (2004), specific brain areas have now been identified for their CNS AVP release patterns: preferentially dendritic (PVN, SON, amygdala) and preferentially axonal (septum, hippocampus).
Centrally released AVP is known to be involved in a wide range of behaviors including repetitive behaviors and emotionality. Thus, the behavioral deficits reported in the present studies suggest that AVP levels are capable of being disrupted in the brain.
Rather than controlling syntax like dopamine, AVP has been shown to be involved with the frequency of grooming bouts and chains (reviewed by Spruijt et al., 1992).
Vasopressin administered intracerebroventricularly to mice provoke a dose dependent grooming response, inducing grooming and scratching (Lumley et al., 2001). It would not be expected that the present study would reveal elevated frequency of grooming chains in the PCB exposed animals, as this “super-stereotypy” would suggest high dopamine levels (Berridge et al., 2005), not characteristic of the dose and combination of congeners used. Future use of the PCB exposed rat as a model for ASD should administer a single coplanar congener, such as PCB 77, which would potentially increase dopamine (Seegal et al., 2005) to observe a possible increase in stereotypic grooming behavior more relevant to the ASD population (McDougle et al., 1999). 49
In terms of emotionality, tests of physiological stress such as forced swimming and social defeat, have shown that AVP is released first in the brain, within the amygdala
(Ebner et al., 2002) and PVN (Wotjak et al., 1996), with no change in systemic plasma
AVP concentration (Wotjak et al., 1996). Landgraf and Neumann (2002) suggest AVP is released first within the brain in response to an emotional stressor and seems to be involved in the generation of passive rather than active coping strategies.
Despite central AVP levels being unchanged in systemic circulation, an animal model of affective and motor dysfunction should evaluate differences in vasopressin synthesis and release within the brain. Specifically relevant to autism, if central AVP- synthesizing neurons are affected, such as the bed nucleus of the stria terminalis (BnST) and the lateral septum, this would affect males more than females. This is because cells of the BnST and the lateral septum are androgen dependent and markedly more abundant in males. In addition to being sexually dimorphic, vasopressin is a strong candidate for childhood disorders because its receptors are developmentally regulated and expressed more in the immature brain (reviewed by Insel et al., 1999).
Clinical Implications: A Possible Animal Model of Autism
To date, there is still no consistent neurochemical, neurophysiological, or
neuroanatomical marker that can be used to identify and diagnose patients with ASD
(Santangelo and Tsatsanis, 2005). Current diagnoses and treatments have had to rely soley on behavioral assessment (Lim et al., 2005). Given the complex nature of autism
spectrum disorder, it is difficult to comprehensively investigate the underlying
neurobiology using only human patients (Lim et al., 2004). Much can be learned by 50 studying the neuroendocrinology of animal models in parallel. Many epidemiological studies have not controlled for key variables: medical history, medication use, environment, demographics, diagnostic criteria, and most importantly, case composition.
(Hollander 2003). Additionally, clinical research has been limited by small sample sizes and limited numbers of case studies. While a rodent model cannot replicate ASD, fundamental symptoms can be approximated for the purposes of testing theories about the biochemical causes of the autistic condition (Crawley, 2004).
The importance of data and implications for an adequate animal model can be expanded to the clinical setting for comparison. Though establishment of a serum neurobiomarker would have been a major advance towards differentiating affective disorders, vasopressin still stands as a strong candidate for dysregulation in autism (Lim et al., 2004; Carter, 2005). The studies discussed in this thesis demonstrate that PCB exposure is capable of causing disruption to similar domains affected in the ASD population (Table 14). Specifically, the different results in animals given 12.5 ppm and
25 ppm PCB may allow use of this system to serve as a model to observe a range of behavioral severity much like that seen in the broad autistic phenotype. Since PCB compounds are such ubiquitous environmental endocrine disruptors capable of affecting the developing brain (London and Etzel, 2000), the present study encourages further investigation into the possibility of exogenous contaminants like PCB as potential environmental triggers for ASD.
51
Table 14: PCB Exposure as a Possible Animal Model for ASD. Clinical ASD Investigations carried out in the PCB-exposed Diagnosis rodent model
b c Social Behavioral paradigms: social port , conditioned odor preference , play behaviorb, social recognitiond
Hormone assays: oxytocind and vasopressina
a a a Motor Behavioral paradigms: open field , hang test , negative geotaxis , righting reflexa, fixed action patternsa (grooming syntax), T-maze reversala
e Communication Behavioral paradigms: ultrasonic vocalizations aPresent Studies bPreliminary unpublished findings cCromwell et al., 2007 dJolous-Jamshidi, 2007 eMcFarland et al., 2007
52
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APPENDIX I: OPEN FIELD ACTIVITY
Mean Horizontal Crosses. Condition PND 14-16 PND 28-32 PND 60-64
Control 61.4 ± 5.8 61.8 ± 5.4 100.9 ± 6.0 n= 16 n= 12 n= 16 12.5 ppm 54.4 ± 8.9 77.0 ± 7.1 80.7 ± 4.5 a n= 24 n= 28 n= 16 25 ppm 58.9 ± 12.7 71.1 ± 8.2 72.2 ± 4.0 a n= 22 n= 16 n= 16 a Significantly different from control group (p<0.05) Values represent mean horizontal score ± standard error of the mean
Mean Rear Count. Condition PND 14-16 PND 28-32 PND 60-64
Control 16.3 ± 2.4 23.5 ± 4.6 42.7 ± 3.3 n= 16 n= 12 n= 16 12.5 ppm 14.6 ± 2.8 42.7 ± 4.6 a 47.9 ± 3.6 n= 24 n= 28 n= 16 25 ppm 10.3 ± 1.8 36.6 ± 5.3 a 46.6 ± 3.4 n= 22 n= 16 n= 16 a Significantly different from control group (p<0.05) Values represent mean number of rears counted ± standard error of the mean
Mean Rear Time. Condition PND 14-16 PND 28-32 PND 60-64
Control 26.5 ± 2.5 46.7 ± 9.8 73.1 ± 5.6 n= 16 n= 12 n= 16 12.5 ppm 36.9 ± 5.3 88.3 ± 9.8 a 72.2 ± 11.5 n= 24 n= 28 n= 16 25 ppm 28.5 ± 5.5 80.1 ± 12.3 a 86.1 ± 9.6 n= 22 n= 16 n= 16 a Significantly different from control group (p<0.05) Values represent mean time spent rearing ± standard error of the mean
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APPENDIX II: HANG, NEGATIVE GEOTAXIS, RIGHTING
Mean Hang Scores.
Condition PND 14-16 PND 28-32 PND 60-64
Control 70.4 ± 6.0 74.3 ± 5.7 90.8 ± 4.9 n= 26 n= 28 n= 12 12.5 ppm 47.3 ± 4.9 a 67.2 ± 5.4 53.1 ± 5.6 c n= 40 n= 32 n= 32 25 ppm 52.6 ± 6.3 57.4 ± 6.4 b 50.9 ± 6.5 c n= 38 n= 36 n= 22 a Significantly different from control group (p<0.05) b Significantly different from control group (p<0.05) c Significantly different from control group (p<0.05) Values represent mean hang score ± standard error of the mean
Mean Negative Geotaxis Scores.
Condition PND 14-16 PND 28-32 PND 60-64
Control 72.5 ± 7.4 78.0 ± 6.4 83.0 ± 7.2 n= 20 n= 28 n= 10 12.5 ppm 55.4 ± 6.8 47.0 ± 6.4a 67.2 ± 6.1 n= 40 n= 32 n= 32 25 ppm 64.5 ± 5.6 41.1 ± 6.5a 63.6 ± 7.5 n= 32 n= 36 n= 22 a Significantly different from control group (p<0.05) Values represent mean hang score ± standard error of the mean
Mean Developmental Righting Reflex in seconds.
Condition PND 14-16 PND 28-32 PND 60-64
Control 1 ± 0.0 1 ± 0.0 1 ± 0.0 n= 26 n= 26 n= 10 12.5 ppm 1.02 ± 0.0 1 ± 0.0 1 ± 0.0 n= 32 n= 32 n= 32 25 ppm 1.1 ± 0.0a 1.01 ± 0.0 1 ± 0.0 n= 30 n= 36 n= 22 Values represent mean righting time ± standard error of the mean a Significantly different from control group (p<0.05)
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APPENDIX III: GROOMING
Mean Total Grooming/ Rat Not Significantly Different Among Treatment Groups.
Condition PND 14-16 PND 28-32 PND 60-64
Control 3.08 ± 0.4 2.39 ± 0.2 2.53 ± 0.6 n= 24 n= 23 n= 15 12.5 ppm 3.34 ± 0.3 2.91 ± 0.3 3.34 ± 0.2 n= 32 n= 32 n= 35 25 ppm 2.57 ± 0.2 3.15 ± 0.4 2.79 ± 0.4 n= 30 n= 34 n= 29 Values represent mean total grooming/rat ± standard error of the mean
Mean Chains Initiated/ Rat Not Significantly Different Among Treatment Groups.
Condition PND 14-16 PND 28-32 PND 60-64
Control 1.96 ± 0.2 2.13 ± 0.2 2.40 ± 0.6 n= 24 n= 23 n= 15 12.5 ppm 2.78 ± 0.3 2.41 ± 0.2 3.14 ± 0.3 n= 32 n= 32 n= 35 25 ppm 2.03 ± 0.2 2.85 ± 0.3 2.28 ± 0.3 n= 30 n= 34 n= 29 Values represent mean chains initiated/rat ± standard error of the mean
Percent Complete Perfect Chain Grooming
Condition PND 14-16 PND 28-32 PND 60-64
Control 25.9 % ± 0.05 87.7 % ± 0.04 90.3 % ± 0.07 n= 24 n= 23 n= 15 12.5 ppm 26.1 % ± 0.05 79.4 % ± 0.07 88.5 % ± 0.04 n= 32 n= 32 n= 35 25 ppm 0 % ± 0 a 80 % ± 0.08 75 % ± 0.08 n= 30 n= 34 n= 29 Values represent mean % complete ± standard error of the mean a Significantly different from control group (p<0.05)
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Percent Incomplete Chain Grooming.
Condition PND 14-16 PND 28-32 PND 60-64
Control 54.5 % ± 0.09 12.3 % ± 0.04 3 % ± 0.02 n= 24 n= 23 n= 15 12.5 ppm 58 % ± 0.07 7.8 % ± 0.39 7 % ± 0.35 n= 32 n= 32 n= 35 25 ppm 93 % ± 0.04a 10 % ± 0.04 11% ± 0.05 n= 30 n= 34 n= 29 Values represent mean % incomplete ± standard error of the mean a Significantly different from control group (p<0.05)
Percent Complete Imperfect Chain Grooming Not Significantly Different Among Treatment Groups.
Condition PND 14-16 PND 28-32 PND 60-64
Control 11.1 % ± 0.04 7.9 % ± 0.03 3.6 % ± 0.02 n= 24 n= 23 n= 15 12.5 ppm 7.9 % ± 0.03 5 % ± 0.03 1.2 % ± 0.01 n= 32 n= 32 n= 35 25 ppm 3.6 % ± 0.02 4 % ± 0.03 0 % ± 0.0 n= 30 n= 34 n= 29 Values represent mean % imperfect ± standard error of the mean
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APPENDIX IV: T-MAZE
Mean Learning Days:
Treatment Group # Learning Days Required
Control 1.60 ± 0.15 n=26
12.5 ppm 2.1 ± 0.18 a n=18
25 ppm 3.5 ± 0.25 a n=25 Values represent mean days for learning ± standard error of the mean a Significantly different from control group (p<0.05) b Significantly different from control group (p<0.05)
Mean T-maze Reversal Scores.
Treatment Group % Correct Reversal Control 77 ± 2.6 n=26 12.5 ppm 66 ± 3.0 a n=18 25 ppm 41 ± 2.8 a n=19 Values represent mean % correct in reversal task ± standard error of the mean a Significantly different from control group (p<0.05)
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APPENDIX V: VASOPRESSIN CONCENTRATIONS
Mean Circulating Serum Vasopressin Concentrations in 29-Day Old Male Rats Not Significantly Different Among Treatment Groups.
Treatment Group Vasopressin Concentration (pg/ml) Control 2.01 ± 1.65 n=26 12.5 ppm 2.01 ± 2.12 n=18 25 ppm 2.02 ± 1.73 n=25 Values represent mean concentration ± standard error of the mean